Mammalian development begins with fertilization, followed by multiple cell cleavage events and the transition from maternal to zygotic transcription. All these events work in concert to establish the first and thus highly significant developmental event, lineage differentiation. The first step in the establishment of different lineages in the pre-implantation embryo gives rise to the trophectoderm (TE), the precursor for extraembryonic structures such as the placenta and yolk sac, and the inner cell mass (ICM), which becomes the embryo proper. While the regulatory interactions and morphological contributions of transcription factors such as Oct3/4, Nanog, Sox2, Tead4, and Cdx2 have been extensively studied, surprisingly little is known about the roles of secreted growth factors during these early stages of development. The presence of bone morphogenetic proteins (BMPs) in these early mouse embryonic stages suggests likely functional roles for BMP signaling in lineage differentiation. This project will determine the significance of BMP signaling in pre- implantation stage embryos by elucidating the spatiotemporal emergence and functional role of BMP signaling. Also, this projects aims to identify molecular interactions between BMPs and lineage specification transcription factors vital for the first lineage differentiation. Through a highly sensitive image segmentation quantification method, immunofluorescence detection and quantification of phosphorylated Smad1/5/8 will denote active BMP signaling in pre-implantation embryos. Loss of function experiments will be used to determine if BMP signaling activity impinges on two major transcriptional networks that control lineage specification during blastulation: the pluripotent regulatory motifs of Oct4/Nanog/Sox2 and the TE committing networks of Tead4, Cdx2, and Gata6. Direct evidence for the biological role of BMP signaling in pre-implantation mammalian embryonic development provides a foundation for understanding novel regulatory interactions between transcriptional regulators and signaling morphogens. This understanding benefits multiple health related disciplines such as stem cell therapeutics, cancer biology, and reproductive medicine.